Botulinumneurotoxinsareafamilyofsevenbacterialtoxins(BoNT/A-G).MembersoftheBoNT family have been widely utilized for treating a growing list of medical conditions. During our previous funding cycle, we carried out the first comprehensive investigation of the effect of BoNTsonsurvivalofneurons.Weidentifiedtwoofthetoxins,BoNT/CandBoNT/E,thatinduce death of neurons. We further established that neuronal death is due to blockage of a plasma membrane recycling process by BoNT/C and E. These findings open a new line of inquiry on toxinbiologyandrevealanovelmembranerecyclingprocessessentialforneuronsurvival. Followingthesefindings,wefurtherfoundthatamyloidprecursorprotein(APP)isamajorcargo ofthemembranerecyclingprocessblockedbyBoNTs.Hereweproposemechanisticstudiesto elucidate this essential membrane recycling process at the molecular level. We will also utilize BoNTs as a novel tool to address key questions about APP recycling/processing and explore the potential role of APP in BoNT action at nerve terminals. Finally, our current studies established that SNAP-25, which is cleaved by BoNT/A, C, and E, is essential for neuron survival and APP recycling. This finding raised a significant safety concern regarding long-term useofthemajortherapeutictoxinBoNT/A,whichpromptedustodevelopalternativetherapeutic toxinsthroughproteinengineering. Our proposed studies will provide a mechanistic understanding of the effect of BoNTs on survival of neurons and may yield novel insights into APP biology. They will also address concerns about the long-term safety of current therapeutic toxins and aim to develop a new generationoftherapeutictoxinswithimprovedefficacyandsafetyinhumans.